Biz bu protokolü gelişmekte olan yardım için Simon Alberti teşekkür ederim. Bu çalışma Ulusal Sağlık Enstitüsü (GM25874), Howard Hughes Tıp Enstitüsü Investigatorship (SL) ve Ulusal Bilim Vakfı predoctoral eğitim bursu (RH) bir hibe ile desteklenmiştir.

<html> Bölüm 1: jel hazırlanması <ol><li> Jel döküm tepsi birleştirin. Standart donanım yatay DNA elektroforezi için kullanılabilir. Çok sayıda numune için, 20 cm kadar dört adet 50-iyi tarak x 24 cm döşeme hazırlar. Bu leke görüntüsünü bozabilir levha, çizik yoktur emin olun. </li><li> 1X TAE% 1.5 agaroz çözeltisi (orta veya yüksek jel gücü, düşük EEO) oluşturun. Hacmi tamamen daldırın tarak dişleri için yeterli olmalıdır tespiti en üst düzeye çıkarmak için mümkün olduğunca çok örnek yüklemek isteyeceksiniz. Mikrodalga karışımı agaroz tamamen eriyene kadar. </li><li> Hızla% 10 oranında hisse senedi% 0,1 SDS ekleyin. Çalkalanarak karıştırılır. Bu adım sırasında bazı agaroz katılaşır varsa, sıcak bir tabak kullanılarak çözülür ve kaynar önlemek için dikkatli olun. </li><li> Çözüm döküm tepsiye dökün. Daha sonra transferi etkileyebilecek herhangi bir kabarcıkları dışarı komisyon bir tarak kullanın. </li><li> Sonra jel seti, tarak kaldırmak jel ve jel tankının içine yerleştirin. Tamamen daldırın 1X TAE jel SDS% 0.1 içeren. </li></ol> Bölüm 2: numunelerin hazırlanması <ol><li> Maya lizatları yüksek verimlilik analizi için, bir gecede 96-iyi blokların hızlı ajitasyon yetiştirilen 2 ml kültürleri ile başlar. Bu durumda, her kültürün ilgi eksprese bir protein. Düşük bolluk proteinleri analiz ederken, büyük kültür hacimleri kullanılması gerekir </li><li> Hasat hücreler santrifüj yoluyla oda sıcaklığında 5 dakika boyunca 2000 RCF. </li><li> Su ve santrifüj hücrelerin tekrar süspanse edin. </li><li> 1 ml spheroplasting çözelti içinde süspanse edin. Yaklaşık 30 dakika boyunca inkübe 30 ° C (Mikroskop ile spheroplasting verimliliği onaylamak). </li><li> Oda sıcaklığında 5 dakika boyunca 800 RCF santrifüjleyin. Tamamen süpernatant kaldırmak. </li><li> 100 ml lizis tamponu pelet spheroplasts tekrar </li><li> 2 dakika için yüksek hızlı bant ve vorteks blok örtün. </li><li> 2 dakika süreyle 4000 RCF Pelet hücre kalıntıları vardır. </li><li> Dikkatle yeni bir konteyner, örneğin, bir 96-PCR plate supernatant çıkarın. </li><li> İsterseniz, lizatları protein konsantrasyonu belirler. </li><li> 1X numune tamponu içeren lizatları üretmek için numuneler 4X numune tamponu ekleyin. Oda sıcaklığında 5 dakika inkübe edin. </li><li> Jel Yük. İsterseniz, örnek hacmi yarısı kaydetmek ve SDS-PAGE analizi için kaynatın. Transfer ölçüde daha sonra izlemek için, bir ön-lekeli SDS-PAGE işaretleme içerir. Ayrıca, çok büyük proteinleri (örneğin, tavuk pektoralis özü) oluşan bir moleküler ağırlık belirteci çözülmesi kompleksleri boyutlarını tahmin etmek için kullanılabilir. </li><li> Boya ön jel sonundan itibaren yaklaşık 1 cm ulaşır kadar alçak gerilim (≤ 3 V / cm jel uzunluğu) çalıştırın. Bu birkaç saat sürebilir. Jel serin kalması çok önemlidir, aksi halde düşük molekül ağırlıklı protein (örneğin, monomerler) difüzyon tespit sınırı. </li></ol> Bölüm 3: Transfer <ol><li> Nitroselüloz, jel gibi aynı boyutta bir parça kesin. </li><li> GB004 20 adet ve 8 adet GB002 blot kağıt, jel gibi aynı boyutta kesin. Kes bir fitil olarak kullanılmak üzere ek bir parça GB004 jel yaklaşık 20 santimetre daha geniş yapmak. </li><li> 1X TBS nitroselüloz, fitil, 4 adet GB002, bırakın. </li><li> 20 adet kuru GB004, daha sonra 4 adet kuru GB002, daha sonra tek parça ön ıslak GB002: Bir plastik kap, aşağıdaki gibi bir yığın kağıtları bir araya. Nitroselüloz bu yığının üstüne yatırın. </li><li> Aşırı çalışan tampon kaldırmak için su döküm tepsi kısaca jel durulayın. Sonra, dikkatle jel yığının üzerine tepsi kapalı slayt başlar. Tepsiyi kapalı jel sürme, gerektiğinde TBS ile membran söndürmeye. Ekstra tampon jel altında sıkışmış olmanın kabarcıkları önlemeye yardımcı olur. Bu amaçla transfer pipet iyi çalışır. </li><li> Jel yığınına taşındıktan sonra, kabarcıklar iyice kontrol edin. Herhangi biri mevcutsa, jel kenar kaldırın ve kabarcıkları dışarı çalıştı kadar tampon yeniden uygulayın. </li><li> Jel üstünde kalan üç önceden ıslatılmış GB002 adet koyun. Yığının üst kısmında bir pipetle iyice haddeleme tüm katmanları arasındaki kapsamlı temas olun. </li><li> TBS içeren iki yükseltilmiş tepsiler Kanat transfer yığını. Öncesi ıslak fitil fitil ya sonunda TBS batmış olduğunu yığını karşısında Örtüsü. </li><li> Ekstra ağırlık taşıyan (örneğin, 500 ml şişe su) ek bir plastik tepsi ile bir araya transferi yığını örtün. </li><li> En az üç saat veya gece boyunca devam transferine olanak sağlar. </li><li> Aktarımdan sonra, membran standart Western blot tarafından işlenebilir. </li></ol> Spheroplasting Çözüm 1,2 M D-sorbitol 0.5 mM MgCl 2 20 mM Tris, pH 7.5 50 mM BME (taze eklemek) 0.5 mg / ml Zymolyase 100T (taze eklemek) coration: underline &quot;&gt; Lizis Tampon 100 mM Tris 7.5 50 mM NaCl 10 mM BME (taze eklemek) proteaz inhibitörleri (taze eklemek) 4X Örnek Arabelleği 2X TAE % 20 gliserol % 8 SDS tercihinize Bromophenol Mavi

<ol>
	<li>Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+[PSI+]+prion+aggregates+are+formed+by+small+Sup35+polymers+fragmented+by+Hsp104.">Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104.</a> J. Biol. Chem. 278, 49636-49643 (2003).</li><li>Alexandrov, I. M., Vishnevskaya, A. B., Ter-Avanesyan, M. D., Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+and+Propagation+of+Polyglutamine-based+Amyloids+in+Yeast:+TYROSINE+RESIDUES+ENABLE+POLYMER+FRAGMENTATION.">Appearance and Propagation of Polyglutamine-based Amyloids in Yeast: TYROSINE RESIDUES ENABLE POLYMER FRAGMENTATION.</a> J. Biol. Chem. 283, 15185-15192 (2008).</li><li>Allen, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperones+as+modulators+of+prion+life+cycle:+novel+effects+of+Ssa+and+Ssb+on+the+Saccharomyces+cerevisiae+prion+[PSI+].">Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].</a> Genetics. 169, 1227-1242 (2005).</li><li>Aron, R., Higurashi, T., Sahi, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J-protein+co-chaperone+Sis1+required+for+generation+of+[RNQ+]+seeds+necessary+for+prion+propagation..">J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation..</a> The EMBO journal. 26, 3794-3803 (2007).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+amyloid+aggregates+using+agarose+gel+electrophoresis.">Analysis of amyloid aggregates using agarose gel electrophoresis.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Borchsenius, A. S., Muller, S., Newnam, G. P., Inge-Vechtomov, S. G., Chernoff, Y. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+variant+maintained+only+at+high+levels+of+the+Hsp104+disaggregase.">Prion variant maintained only at high levels of the Hsp104 disaggregase.</a> Current Genetics. 49, 21-29 (2006).</li><li>Salnikova, A. B., Kryndushkin, D. S., Smirnov, V. N., Kushnirov, V. V., Ter-Avanesyan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsense+suppression+in+yeast+cells+overproducing+Sup35+(eRF3)+is+caused+by+its+non-heritable+amyloids.">Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids.</a> J. Biol. Chem. 280, 8808-8812 (2005).</li><li>Tank, E. M., Harris, D. A., Desai, A. A., True, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+repeat+expansion+results+in+increased+aggregation+and+reveals+phenotypic+variability.">Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability.</a> Mol. Cell. Biol. 27, 5445-5455 (2007).</li><li>Douglas, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone-dependent+amyloid+assembly+protects+cells+from+prion+toxicity.">Chaperone-dependent amyloid assembly protects cells from prion toxicity.</a> Proc. Natl. Acad. Sci. USA. 105, 7206-7211 (2008).</li><li>Nagy, B., Costello, R., Csako, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downward+blotting+of+proteins+in+a+model+based+on+apolipoprotein(a)+phenotyping.">Downward blotting of proteins in a model based on apolipoprotein(a) phenotyping.</a> Analytical Biochemistry. 231, 40-45 (1995).</li></ol>

SDD-YAŞ ilk Kryndushkin ve arkadaşları tarafından rapor edilmiştir 1, SDS-dirençli kompleksleri çalışma [PSI +] maya prion, ve o zamandan beri hem prion ve prion olmayan agrega 2-9 okuyan yaygın kullanımı bulmuştur. Ancak, bir agaroz jel bir zar aşağıdaki elektroforez, proteinlerin transferi sorunlu ve çarpık bir leke görüntü 5 neden olabilir . Ayrıca, en sık kullanılan batık electroblotting tekniği jel boyutu için pratik sınırlamalar tanıtır ve böylece işlenebilir örneklerin sayısı. Biz aşağı kılcal transfer 10, jel nitroselüloz membran proteinleri aktarmak için bir yığın kuru blot kağıtları kullanan basit bir prosedür kullanılarak bu sorunları ele alınmıştır. Kılcal transfer bozulmasını engeller ve büyük jeller kolay işlenmesine olanak verir. SDD-YAŞ kullanarak önce dikkate alınması gereken bir kaç şey vardır. Ham örnekler için (örneğin, lizatları), spesifik proteinlerin İmmuno gerekli. SDD-AGE tamamen ilgi protein kompleksleri doğasını değildir, protein (lar) tespit edilmesi, amiloidojenik bölgenin dışında bir epitopu etiketi taşımalıdır. Lizatları genellikle normal bir SDS-PAGE olacağı gibi iki önemli farklılıklar hazırlanmış olabilir. İlk olarak, proteoliz tarafından bozulmasını önlemek için artan dikkat edilmelidir. Burada kullanılan kısmen denatüre koşulları proteazlar inaktive etmek için yeterli değildir ve de proteoliz hedef proteinler daha duyarlı yapabilirsiniz. En az iki kat önerilen konsantrasyonda tam bir proteaz inhibitörü kokteyl kullanın. İkincisi, numunelerin ısıtma kaçınılmalıdır. Bir all-monomer negatif kontrol isteniyorsa, örneğin yüksek molekül ağırlıklı türler monomerik protein en amiloidleri geri kovalent modifikasyonlar, 10 dakikalık bir inkübasyon 95 ° C kullanılabilir nedeniyle olmadığını onaylamak için.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>zymolyase 100T</td>
<td>&nbsp;</td>
<td>Seikagaku America</td>
<td>120493-1</td>
<td>&nbsp;</td>
<tr>
<td>Halt Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>78429</td>
<td>&nbsp;</td>
<tr>
<td>Hybond-C extra nitrocellulose</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN303E</td>
<td>&nbsp;</td>
<tr>
<td>GB004 blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>10427926</td>
<td>&nbsp;</td>
<tr>
<td>GB002 (3MM Chr) blotting paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-917</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (UltraPure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15510-027</td>
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screening for amyloid aggregation by semi-denaturing detergent-agarose gel electrophoresis

Amiloid agregasyonu conformationally olan pek çok protein misfolding patolojiler ve temelini prionlar bulaşıcı özellikleri, kendi kendine şablonu daha aşağı organizmalardan yararlı rolleri olduğu düşünülmektedir proteinler. Ile ilişkili. Amiloidleri çıkılmazlık ve yapısal heterojenite nedeniyle çalışma için herkesin bildiği gibi zor olmuştur. Ancak, boyutu ve deterjan çıkılmazlık dayalı amiloid polimerler çözünürlüğü mümkün hale olmuştur Yarı denatüre Deterjan Agaroz Jel Elektroforezi (SDD-AGE). Bu tekniğin yaygın kullanımı amiloid konformasyonel türevlerini tespit edilmesi ve karakterizasyonu için bir bulgudur. Burada, roman prionlar ve diğer amiloidojenik proteinler için ekranlar gibi büyük ölçekli uygulamaları, kullanımı kolaylaştıran bu tekniğin bir uyum göstermektedir. Daha fazla güvenilirlik için yeni SDD-AGE bir yöntem kılcal transferi kullanır ve kullanım kolaylığı, ve herhangi bir büyüklükteki jel yer sağlar. Böylece, hücre veya saflaştırılmış proteinlerin hazırladığı numuneler çok sayıda, etiketli proteinlerin SDS-çözünmez conformers varlığı için aynı anda işlenebilir.

Watch this Scientific Journal Video about Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis at JoVE.com

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

SDD-YAŞ hücrelerde polimerler gibi amiloid algılama ve karakterizasyonu için yararlı bir tekniktir. Burada, bu tekniğin büyük ölçekli uygulamalarda mükellef kılar bir uyum göstermektedir.

Yarı denatüre Deterjan-Agaroz Jel Elektroforez Amiloid Toplama taraması

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

screening-for-amyloid-aggregation-semi-denaturing-detergent-agarose

Research

JoVE Journal

Biology

26.7K Görüntüleme.  Whitehead Institute for Biomedical Research. SDD-YAŞ hücrelerde polimerler gibi amiloid algılama ve karakterizasyonu için yararlı bir tekniktir. Burada, bu tekniğin büyük ölçekli uygulamalarda mükellef kılar bir uyum göstermektedir.

Video: Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Denatüre Gradient Jel Elektroforezi (DGGE)

Biyoloji

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denatüre Üre Poliakrilamid Jel Elektroforez (Üre SAYFA)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Hücresel lizatları Multiprotein Kompleksleri Analizi Mavi Native Poliakrilamid Jel Elektroforez (BN-PAGE)

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

Ticari kasetleri yeniden kullanılmasıyla Dökme ve Protein Jel Koşu

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

DNA kırıntıları Ayrılma Agaroz Jel Elektroforezi

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Musin Agaroz jel elektroforezi: Yüksek molekül ağırlıklı glikoproteinler için Western Blot


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal mikroskobu ortaya hücre yüzey reseptör toplama görüntü korelasyon spektroskopisi ile

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.

This protocol demonstrates the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis. Here the method applied to cultured human cells is described.

Mitokondrial solunum zinciri kompleksleri kültürlü insan hücrelerinde mavi yerli Polyacrylamide Jel Elektroforez ve Immunoblotting kullanarak Analizi

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb ...